The aim of this project is to understand the nature of the rod region of dystrophin. Although this region makes up the bulk of dystrophin's sequence, the much smaller terminal globular domains are often considered more important since the mediate dystrophin's interactions with other cellular components. In many cases, shortened versions of dystrophin in which part of the rod region is removed - the so-called mini- dystrophins - are being studied as possible gene therapy replacements. However, substantial context effects have been seen in animal models, in which slightly differently constructed mini-dystrophins involving different rod modifications have had markedly different efficacies. As well, it has recently been demonstrated that the processing of the dystrophin gene naturally produces various deletions (at least at the RNA level) though exon skipping, which may be therapeutically harnessed. However, the biophysical and biochemical construction of the dystrophin rod is not well understood. It is traditionally thought of as composed of a number of repetitive motifs with a few interspersed so-called hinge regions. However, I have shown that the manner in which these regions interact is heterogeneous - some motifs appear independent of their neighbors, while others are not, exhibiting strongly cooperative structural interactions. This has obvious implications for editing, since editing in a cooperative region can have distal effects. As well, normal RNA splicing events appear to produce natural deletion variants via exon skipping, but the pattern is perplexing, in that most edits do not occur near the junctions of these repeat motifs (as have been nearly exclusively employed in man-made deletions), but midway through, resulting in novel, hybrid motifs. All of these factors contribute to the unpredictability of producing functional edited dystrophin variants. We seek to produce a biophysical and biochemical map of the domain structure of the dystrophin rod, thereby providing a rational basis to edit this molecule, and a better understanding of the putative variants produced by normal differential RNA processing. This work seeks to understand the structure of the rod region of dystrophin (the protein defective in Duchenne Muscular Dystrophy), which is the largest component of this protein, and is where most mutations responsible for DMD occur. This rod is composed of many repetitive regions at both the protein and DNA levels, and the manner in which it is assembled suggests that it can be edited to ameliorate these defects. In many therapeutic strategies under development, edited and shortened versions of this rod are envisioned;and we will determine how to produces these shorted regions while still maintaining stability of the protein as a whole.